H-Y antigen

ABSTRACT

The present invention relates to a peptide which is immunologically recognizable as a T cell epitope of the minor histocompatibility antigen H-Y. The peptide comprises amino acid sequence SPSVDKARAEL (SEQ ID NO:1), or FIDSYICQV (SEQ ID NO:2). The peptide is obtainable from the minor histocompatibility antigen H-Y. Providing a toxic moiety to the peptide eliminates T Cells having specific binding affinity for the peptide. The peptide induces tolerance for transplantations when administered to H-Y-negative recipients.

This application is a National Stage application under 35 U.S.C. §371 ofPCT/NL96/00307.

The invention relates to the field of immunology, in particular to thefield of cellular immunology.

It is also concerned with the area of organ transplantation, grafting oftissues or cells, especially bone marrow and possible immunologicalreactions caused by transplantation and/or grafting andbloodtransfusion.

Since the invention concerns a sex-related proteinaceous material,encoded in nature by a sex-related gene, the invention also relates tothe areas of sex linked congenital aberrations, of embryonic selectiontechniques, in vitro fertilization techniques, vaccination and in ovovaccination.

Bone marrow transplantation (BMT), one of the areas the invention isconcerned with and the area from which the present invention originates,finds its application in the treatment of for instance severe aplasticanaemia, leukaemia and immune deficiency diseases.

In the early days of this technique many transplants failed throughrejection of the graft by the host. Transplants that did succeed,however often led to an immune response by lymphocytes present in thegraft against various tissues of the host (Graft versus Host Disease(GvHD)). It is now known that the GvHD response is mainly due to thepresence of major H antigens which present a transplantation barrier.Therefor it is now routine practice to graft only HLA-matched materials(either from siblings or unrelated individuals) resulting in a muchimproved rate of success in bone marrow transplantation. However,despite this improvement, as well as improvements in pretransplantationchemotherapy or radiotherapy and the availability of potentimmunosuppressive drugs, about 20-70% of the treated patients stillsuffer from GvHD (the percentage is age and bone marrow donordependent). To avoid GvHD it has been suggested to remove the cells(mature T cells) causing said reaction from the graft. This howeveroften leads to graft failure or to recurrence of the original disease.The cells responsible for GvHD are also the cells which often reactagainst the original aberrant cells in for instance leukaemia (Graftversus Leukaemia response).

Since BMT is nowadays only carried out with HLA matched grafts, the GVHDwhich still occurs must be caused by another group of antigens. It isvery likely that the group of so called minor H antigens (mHag), whichare non-MHC encoded histocompatibility antigens (unlike the major Hantigens) are at least partially responsible for the remaining incidenceof GvHD.

mHag's have originally been discovered in congeneic strains of mice intumor rejection and skin rejection studies. In mice, the use of inbredstrains has shown that mHag are encoded by almost 50 differentallelically polymorphic loci scattered throughout the genome (24). Inhumans, mHag have been shown to exist, although their overall number andcomplexity remains uncertain. One of the better known, thoughunidentified minor histocompatibility antigens is the H-Y antigen. Inthe first report of H-Y as a transplantation antigen Eichwald andSilmser observed that within two inbred strains of mice, most of themale-to female skin grafts were rejected, whereas transplants made inother sex combinations nearly always succeeded (1). The term H-Y antigenwas introduced by Billingham and Silvers (2) because the male specificantigen can function as a classical transplantation antigen responsiblefor homograft rejection.

Alloimmunity to human H-Y was first demonstrated in a female patientwith aplastic anaemia who was given bone marrow from her HLA-identicalbrother. After a period of transient chimaerism the graft was rejected.At this time after grafting her lymphocytes showed unambiguously strongMHC restricted cytotoxic T cell (CTL) responses specific for male HLA-A2positive target cells (3,4). The clinical case not only evidenced thatH-Y can function as a transplantation barrier in man as well, but alsothat the recognition of the human male specific minor Histocompatibilityantigen (mHag) was MHC restricted (4). The clinical relevance of H-Y asalloantigen is demonstrated especially in bone marrow transplantation(BMT) where sex-mismatch is one of the risk factors associated withrejection (3,4,5) or Graft-versus-Host-Disease (6,7). Sensitization tothe H-Y antigen extends to organ transplantation (8-11),bloodtransfusion (12) and pregnancy (13), wherein MHC restricted T cellresponses to the mHag H-Y in association with different MHC moleculesare observed. To understand the impact of mHag H-Y on the outcome oforgan—and bone marrow grafting we earlier studied its tissuedistribution. CTL mediated lysis of tissue-derived cell and culturedcell lines of several human tissues demonstrated an ubiquitousexpression (11,14,16).

In search for the biological function of the gene encoding the mHag H-Y,our earlier studies analyzing lymphocytes from sex chromosomalabnormalities with our HLA restricted H-Y specific CTL clones revealedthat absence of the mHag H-Y correlated with the XO and XX karyotype(17). Subsequent studies combining DNA, and functional expression withour CTL clones analyzing lymphocytes from individuals with Y chromosomaldeletions, assigned the H-Y gene encoding the mHag H-Y to a portion ofinterval 6 (18), to a region covering the proximal segment of the Yqeuchromatin, on the long arm of the Y chromosome (19).

Besides the role of H-Y as transplantation antigen, the human Y genecontrolling the expression of the mHag H-Y is possibly also functioningas a gene controlling spermatogenesis. Agulnik et al. (20) recentlyidentified a new murine Y chromosome gene, designated Smcy, controllingspermatogenesis as well the expression of the murine male specific mHagH-Y. The Smyc gene appears to be conserved on the Y chromosome in mouse,man and even in marsupials (20). It is notable that recent studies fromour laboratories show recognition of the human HA-2 and H-Y peptides onnon human primates cells, transfected with human class I genes, by ourhuman HA-2 and H-Y specific class I restricted CTL clones (21).

Until recently, little was known about the molecular nature of the mHaggene products. Recent evidence was obtained revealing that thenon-sexlinked human mHag HA-2 represents a short peptide originatingfrom a member of the non-filament-forming class I myosin family (22).However, no information exists on the amino-acid sequence nor on theprotein of the male specific mHag H-Y.

Aiming at the identification of the human H-Y peptide, we used theHLA-B7 restricted CTL clone “5W4” (12). Clone 5W4 originates from afemale aplastic anemia patient who had received multiple transfusions(12,23).

Besides the HLA-B7 H-Y specific CTL clone, we earlier characterizedHLA-A2 as well as HLA-A1 H-Y specific CTL clones (23).

We used a CD8 positive HLA-A2.1 restricted H-Y specific CTL clone,designated “1R35” (23). Besides, we also previously characterized a CD4positive HLA-A2.1 restricted H-Y specific cytotoxic as well asproliferative T cell clone, designated as “R416” (41).

We aimed at identification of the human H-Y peptide recognized by theHLA-A2.1 restricted H-Y specific T cell clones IR35 and R416. The samemethodology as applied for the identification of the HLA-B7 restrictedH-Y peptide was used.

The invention thus provides a (poly)peptide comprising a T-cell epitopeobtainable from the minor Histocompatibility antigen H-Y comprising thesequence SPSVDKARAEL (SEQ ID NO:1) or FIDSYICQV (SEQ ID NO:2) or aderivative of either of these having similar immunological properties.

The two sequences specified are encoded by the SMCY gene. The firstsequence is the one found using the HLA-B7 restricted H-Y specificT-cell clone, The second is the one found using the HLA-A2.1 restrictedclones.

The way these sequences are obtained is described herein. An importantpart of this novel method of arriving at said sequences is thepurification and the choice of the starting material. Said novel methodis therefor also part of the scope of this invention. However, now thatthe sequence is known, it is of course no longer necessary to followthat method, because the peptides can easily be made synthetically, asis well known in the art. Since routine techniques are available forproducing synthetic peptides, it is also within the skill of the art toarrive at analogs or derivatives of the explicitly described peptides,which analogs and/or derivatives may have the same or at least similarproperties and or activity. On the other hand analogs which counteractthe activity of the explicitly described peptides are also within theskill of the art, given the teaching of the present invention. Thereforderivatives and/or analogs, be it of the same or different length, be itagonist or antagonist, be it peptide-like or peptidomimetic, are part ofthe scope of this invention.

A preferred embodiment of the present invention are the peptides withthe sequences SPSVDKARAEL (SEQ ID NO:1) and/or FIDSYICQV (SEQ ID NO:2).This does not imply that other peptides are not suitable. This will fora large part depend on the application and on other properties of thepeptides, which were not all testable within the scope of the presentinvention.

The peptides and other molecules according to the invention find theirutility in that they may be used to induce tolerance of the donor immunesystem in H-Y negative donors, so that residual peripheral bloodlymphocytes in the eventually transplanted organ or the bone marrow, asit may be do not respond to host H-Y material in an H-Y positiverecipient. In this way GvHD may be prevented. On the other handtolerance may be induced in H-Y negative recipients in basically thesame way, so that upon receipt of an organ or bone marrow from an H-Ypositive donor no rejection on the basis of the H-Y material occurs.

For tolerance induction very small doses can be given repeatedly, forinstance intravenously, but other routes of administration may very wellbe suitable too. Another possibility is the repeated oral administrationof high doses of the peptides. The peptides may be given alone, or incombination with other peptides, or as part of larger molecules, orcoupled to carrier materials in any suitable excipients.

Further applications of the peptide or derivatives thereof lie in theprophylactic administration of such to transplanted individuals toprevent GvHD. This can be done with either agonists, possibly incombination with an adjuvant, or with antagonists which may block theresponsible cells. This can be done with or without the concomittantadministration of cytokines.

Furthermore the peptides or antibodies thereto can be used in so called“magic bullet” applications, whereby the peptide or the antibody iscoupled to a toxic substance to eliminate certain subsets of cells.

Diagnostic applications are clearly within the skill of the art. Theyinclude, but are not limited to H-Y typing, detection of geneticaberrancies and the like.

Other therapeutical applications of the peptide include the induction oftolerance to H-Y proteins in H-Y related (auto)immune diseases, such aspossibly in Rheumatoid arthritis. On the other hand they may be used invaccines in H-Y related (auto)immune diseases.

For the sake of illustration a number of applications is cited below.

The H-Y peptide or its derivatives can be used to prevent harmfulreaction of the recipient towards the donor or vice versa; in all formsof transplantation i.e. organs, tissues and bone marrow. Assuming thatresidual donor peripheral blood lymphocytes (PBL)'s in the transplantedorgan could react with and/or against host PBL's and even could causeGvHD, the H-Y peptide could be used to induce tolerance in living organ(kidney, liver, gut, skin) of H-Y negative donors for H-Y positivepatients. In bone marrow transplantation, the H-Y peptide (given aloneor in combination with other peptides) can be used to induce tolerancein the living bone marrow donor. The peptide(s) can be given orally,intravenous or otherwise.

In all forms of organ (including cornea), tissue (including heartvalvesand skin) and bone marrow transplantation with living or cadavericdonors, the H-Y peptide could be used to induce tolerance in H-Ynegative recipients of organ and tissue transplants from H-Y positivedonors. In case of bone marrow transplantation, tolerance must beinduced in female donors for male recipients. The tolerance inductioncan be achieved by clinical application of the H-Y peptidesystematically, i.v., locally, orally, as eye-drops.

The H-Y peptides could act in a non-allelic restricted manner (thuspromiscuous) implicating that its applicability to inducing tolerance isnot restricted to the HLA type of the female donors and femalerecipients and donors.

The H-Y peptides or their derivatives can be applied to generatereagents and/or medicine. They can be used as Graft-versus-Host diseaseand rejection prophylaxis administration to the transplanted individualeither with or without adjuvant of

a) a H-Y peptide

b) H-Y peptide analogues, including left or right turning peptides

c) H-Y peptide antagonists

Usage of the H-Y sequence information to generate, for immunomodulatorypurposes:

a) anti-idiotypic T cells

b) anti-idiotypic B cells

c) human monoclonal antibodies

The H-Y peptides or their derivatives can be used as a marker for sexlinked congenital or other diseases.

They can be used for the generation of a genetic probe enablingscreening for the congenital sex-linked disorders.

The genetic probe can be used for genetic counseling, populationgenetics and pre-natal diagnostic.

The defect can be repaired by genetic engineering.

The peptides and other molecules according to the invention can also beused for the production of anti-conceptive drugs.

Furthermore the peptides and other molecules according to the inventioncan be used for the production of cytotoxic T lymphoctes (CTL) withspecificity for the H-Y sequence.

The H-Y specific CTL can be used for selection of male embryos in Xlinked recessive disorders.

The invented molecules can be applied to generate reagents and/ormedicine for

a) determination of foetal erythrocytes in maternal circulation.

b) intra uterine diagnostics

c) use prior to implantation for in vitro fertilization.

d) determination of chimerism.

Veterinary applications include:

a) embryonic selection.

b) in vitro fertilization.

c) vaccination and in ovo vaccination

d) anti-conception.

On the basis of the peptides described herein genetic probes can beproduced which can be used to screen for the gene encoding the protein.On the other hand such probes may be useful in detection kits as well.On the basis of the peptides described herein anti-idiotypic B cellsand/or T cells and antibodies can be produced. All these embodimentshave been made possible by the present disclosure and therefor are partof the present invention.

The techniques to produce these embodiments are all within the skill ofthe art.

Dose ranges of peptides and antibodies and/or other molecules accordingto the invention to be used in the therapeutical applications asdescribed herein before are usually designed on the basis of rising dosestudies in the clinic. The doses for peptides may lie between about 0.1and 1000 μg per kg bodyweight, preferably between 1 and 10 μg per kgbodyweight.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1.

Reconstitution of the H-Y epitope with HPLC fractionated peptidesextracted from HLA-B7 molecules. (A) HLA-B7 molecules wereimmunoaffinity purified from 2×1010 H-Y positive JY cells. Peptides wereeluted from B7 molecules with 10% acetic acid, pH 2.2, filtered througha 10 kD cut-off filter and fractionated on a C18 reverse phase column.Buffer A was 0.1% heptafluorobutyric acid (HFBA); buffer B was 0.1% HFBAin acetonitrile. The gradient consisted of 100% buffer A (0-20 min), 0to 12% buffer B (20 to 25 min), and 12 to 50% buffer B (25 to 80 min) ata flow rate of 200 μl/min. 60 fractions of 200 μl each were collectedfrom 20 to 80 min. (B) Fractions 28 and 29 from the separation shown in(A) were rechromatographed with the same acetonitrile gradient, butusing trifluoroacetic acid (TFA) instead of HFBA as the organicmodifier. For both panels, 3% of each peptide fractions werepreincubated with 1,000⁵¹Cr-labeled T2-B7 cells at room temperature for2 hours. CTLS were then added at an effector to target ratio of 10 to 1,and further incubated at 37° C. for 4 hours. Background lysis of T2-B7by the CTL in the absence of any peptides was −3% in (A) and −4% in (B);positive control lysis of JY was 75% in (A) and 74% in (B).

FIG. 2.

Determination of candidate H-Y peptide by mass spectrometry combinedwith ⁵¹Cr release assay. HPLC fraction 14 from the separation shown inFIG. 1B was chromatographed with an on-line microcapillary columneffluent splitter as previously described (11,13). One-fifth of theeffluent was deposited into μl of culture media in microtiter platewells for analysis with CTLs as in FIG. 1. The remaining four-fifths ofthe material were directed into the electrospray ionization source, andmass spectra of the peptides deposited in each well were recorded on atriple-quadruple mass spectrometer (Finnigan-MAT, San Jose, Calif.).

(⋄), H-Y epitope reconstitution activity measured as percent specificlysis; (▪), abundance of peptide 1171 measured as ion current at m/z391.

CAD mass spectrum of peptide 1171 after conversion the R residue toomithine. material from second dimension HPLC fraction 14 shown in FIG.1B was treated with 70% hydrazine hydrate for 1 hour. The CAD massspectrum was recorded on the (M+2H)+2 ion at m/z 566.

FIG. 4.

H-Y epitope reconstitution with synthetic peptides. Synthetic peptideswere purified to homogeneity by reverse phase-HPLC on a Vydac C4 column.Purity was established on an analytical RP column and the quantity ofeach peptide was confirmed by comparing the area of the peak with thatof a standard peptide. The identity of the peptides was confirmed bymass spectrometry. ⁵¹Cr release was assayed at an effector to targetratio of 10 to 1 on T2-B7 cells that had been incubated with theindicated concentration of SMCY peptide SPSVDKARAEL (⋄) (SEQ ID NO:1),or SMCX peptide SPAVDKAQAEL (▪) (SEQ ID NO:3).

FIG. 5.

Binding of synthetic peptides to purified HLA-B7. HPLC-purified testpeptides were assayed for the ability to inhibit the binding of theiodinated endogenous B7 peptide APRTYVLLL (SEQ ID NO:5) to purfiedHLA-B7 as previously described (40). (⋄), SMCY peptide SPSVDKARAEL (SEQID NO:1); (▪) SMCX peptide SPAVDKAQAEL (SEQ ID NO:3); (▴), APRTLVLLL(SEQ ID NO:5), an endogenous peptide bound to HLA-B7; (x) LLDVPTAAV (SEQID NO:6), an endogenous peptide bound to HLA-A2.1 as the negativecontrol.

FIG. 6.

HLA-A2 molecules were immunoaffinity purified from 10¹⁰ DM cells.Peptides were eluted according to the methodology as described in legendto FIG. 1.

DESCRIPTION OF THE INVENTION

As with other mHag, the recognition of H-Y by T lymphocytes isMHC-restricted (3, 24, 25), and it has been shown that some H-Y antigensare peptides derived from cellular proteins that are presented on thecell surface in association with MHC class I molecules (26). We havedeveloped a technique for the identification of individual peptides thatare bound to MHC molecules and recognized as antigens by T cells. Bycombining microcapillary liquid chromatography/electrospray ionizationmass spectrometry with T cell epitope reconstitution assays, wepreviously identified peptide antigens recognized by T cells specificfor human melanoma (27), human xenografts (28), and a non-sex-linkedhuman mHag (22). We now report the identification of a peptide antigenrecognized by a human cytotoxic T lymphocyte (CTL) clone that is H-Yspecific and restricted by the class I MHC molecule HLA-B7, as well as apeptide antigen that is recognized by two HLA-A2.1 restricted CTLclones.

To isolate endogenously processed H-Y peptides, HLA-B7 molecules werepurified by affinity chromatography from the H-Y positive, Blymphoblastoid cell line, JY (29). The associated peptides wereextracted in acid and separated from high molecular weight material byultrafiltration as previously described (31), and subsequentlyfractionated by reverse-phase high-performance liquid chromatography(HPLC) (27). Aliquots of each fraction were incubated with HLA-B7positive, H-Y negative T2-B7 target cells in order to assay for theability to reconstitute the epitope recognized by an HLA-B7-restricted,H-Y specific CTL clone, 5W4 (ref. 12). A single peak of reconstitutingactivity was observed (FIG. 1A, fraction 28 and 29), which wasrechromatographed using a different organic modifier. Although a singleactive peak of reconstituting activity was also observed from thisseparation (FIG. 1B, fraction 14, 15 and 16), it still contained morethan 100 distinct peptide species, as assessed by electrosprayionization tandem mass spectrometry.

To identify active H-Y peptides in this mixture, we applied each activefraction separately to a microcapillary HPLC column and split theeffluent following the separation (11): Four-fifths of the effluent wasdirected into the mass spectrometer for analysis, while one-fifth wassimultaneously directed into a 96-well microtiter plate for a subsequentepitope reconstitution assay. The amount of the H-Y sensitizing activityin each well was correlated to signals observed in the mass spectrum,and therefore to the abundance of different peptide species. Bycomparing the profile of H-Y activity and the ion abundance data (FIG.2), we were able to identify an (M+3H)+3 ion at a mass-to-charge ratio(m/z) of 391 (neutral molecular mass=1171), whose abundance correlatedwith the amount of H-Y epitope reconstituting activity. Furtherconfirmation of the importance of peptide 1171 was provided by thedemonstration that a peptide with an identical mass andcollision-activated dissociation (CAD) spectrum was also present inHLA-B7 associated peptides extracted from a second H-Y positive Blymphoblastoid line, DM, but absent from a spontaneous H-Y antigen lossvariant of this cell, DM(−) (33).

Assignment of a complete amino acid sequence to the 1171 peptide fromthe CAD mass spectrum recorded at the 20 fmol level proved difficult dueto the absence of high mass fragment ions containing the amine terminus(b-type ions). A series of single and/or doubly charged fragment ionscontaining the carboxyl terminus (y-type ions) identified the C-terminalresidue as either L or I and the first six amino acids as SPSVDK SEQ IDNO:7. The difference in molecular mass between this partial sequence andthat of the full length peptide suggested the presence of fouradditional residues, for a total length of 11. Since the candidatepeptide existed exclusively in the gas phase as an (M+3H)+3 ion, andunderwent mass shifts of 42 and 84 Da on conversion to the correspondingmethyl ester and acetylated derivative, respectively, two of theremaining residues were assigned as R and either D or E. Only twocombinations of four residues (AREA SEQ ID NO:8 and GRDV SEQ ID NO:9)meet the above criteria and satisfy the missing mass of 427 Da. CADspectra recorded on synthetic peptides suggested that R could not belocated at either position 7 or 10. Data bases were searched forproteins containing peptides with these characteristics, and a sequenceconsistent at 9 out of 11 positions was found in residues 909-919 of theprotein encoded by a gene called XE169 or SMCX (34), which is located onthe X chromosome. A homolog of SMCX, called SMCY, is located on the Ychromosome (20). This protein (35) contains a sequence (residues902-912) that is consistent at 11 out of 11 positions, and has theexpected mass of 1171 Da. A CAD mass spectrum recorded on the naturallyprocessed material after conversion of the R residue to ornithineconfirmed that its sequence was identical to that found in the SMCYprotein (FIG. 3).

In the same manner as described above for the HLA-B7 restricted T-cellclone, the peptide recognized by two HLA-A2.1 T-cell clones wasidentified. In short the HLA-A2.1 restricted H-Y specific T cell cloneR416 recognizes HPLC fraction 34, the HLA-A2.1 restricted H-Y specific Tclone 1R35 recognizes HPLC fractions 36 and 39 (FIG. 6). The amino acidsequence analyses and H-Y reconstitution assays demonstrate that bothHLA-A2.1 restricted H-Y specific T cell clones recognize peptidesequence FIDSYICQV (SEQ ID NO:2) with a m/z ratio of 544 or thecystinylated form of the same peptide with a m/z ratio of 604.

A synthetic peptide corresponding to the 11 residue SMCY sequence(SPSVDKARAEL SEQ ID NO:1) was found to sensitize T2-B7 cells forrecognition by the H-Y specific CTL clone. Half-maximal lysis wasachieved at a peptide concentration of 10 pM (FIG. 4). The correspondingpeptide derived from the sequence of the X chromosomal homolog, SMCX,has substitutions of A for S at position 3 and Q for R at position 8.Although this peptide also was able to sensitize T2-B7 cells forrecognition, comparable levels of killing were only achieved by using a10,000-fold higher peptide concentration. Binding studies showed thatthe concentration of the SMCY peptide that inhibited the binding of aniodinated standard peptide to purified HLA-B7 by 50% (IC50) was 34 nM,while the IC50 for the SMCX peptide was 140 nM (FIG. 5). Thus, thesignificant difference in the ability of the SMCY and SMCX peptides tosensitize targets for T cell recognition is almost entirely due to thefine specificity of the T cell receptor, rather than to differences inMHC binding affinities. The SMCX peptide is also present in naturallyprocessed peptide extracts of HLA-B7, although its abundance is only 25%of that of the SMCY peptide (33). Based on all of this information, weconclude that the peptide epitopes representing the HLA-B7 restrictedH-Y antigen is derived from the protein encoded by SMCY, which is alsotrue for the HLA-A2.1 recognized peptide, also encoded by SMCY.

The location of the SMCY gene and the control of its expression fit wellwith those expected of the H-Y antigen based on previous work. Deletionmapping in humans has placed the HY locus to a portion of interval 6 onthe long arm of the human Y chromosome (18), and SMCY maps to this sameinterval (20). H-Y antigens are expressed ubiquitously in differenttissues (5,15), and expression of SMCY has been detected in all maletissues tested (20). One interesting issue is whether the H-Y epitopepeptides presented by other MHC molecules will also be derived fromSMCY. SMCY and SMCX are 85% identical at the amino acid sequence level,and the SMCX gene is expressed ubiquitously from both the active and theinactive X chromosomes in both mice and human (34,36). Therefore,self-tolerance to SMCX will limit the number of SMCY peptides that couldgive rise to H-Y epitopes in association with different MHC molecules.On the other hand, SMCY contains almost 1500 residues, and the over 200amino acid sequence differences between it and SMCX are scatteredrelatively uniformly throughout its length. Thus, there is the potentialto generate a large number of distinct SMCY-specific peptides as H-Yepitopes. It is still an open question whether the H-Y epitope peptidespresented by other MHC molecules are also derived from SMCY. Geneticmapping of the mouse Y chromosome has suggested at least two and up tofive distinct loci encoding H-Y antigens (37). Interestingly, a murineH-Y epitope restricted by H-2Kk has also been shown to be derived fromthe murine Smcy protein (38). The demonstration that two H-Y epitopesfrom either mouse or human are derived from the same protein makes SMCYthe prime target in searching other H-Y epitopes.

The identification of the protein that gives rise to an H-Y antigenculminates 40 years of uncertainty regarding its origin. However, thefunction of SMCY, as well as the homologous SMCX, remains unclear. Bothproteins share significant sequence homology to retinoblastoma bindingprotein 2, which has been suggested to be a transcription factor (39).Nonetheless, this and other H-Y specific peptides are candidates forimmunomodulatory approaches in bone marrow transplantation. They mayalso form the basis for genetic probes to be used for prenatal diagnosisin sex-linked congenital abnormalities, as well as for investigatingminimal residual disease and chimerism.

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9 1 11 PRT Homo sapiens 1 Ser Pro Ser Val Asp Lys Ala Arg Ala Glu Leu 15 10 2 9 PRT Homo sapiens 2 Phe Ile Asp Ser Tyr Ile Cys Gln Val 1 5 3 11PRT Homo sapiens 3 Ser Pro Ala Val Asp Lys Ala Gln Ala Glu Leu 1 5 10 49 PRT Homo sapiens 4 Ala Pro Arg Thr Tyr Val Leu Leu Leu 1 5 5 9 PRTHomo sapiens 5 Ala Pro Arg Thr Leu Val Leu Leu Leu 1 5 6 9 PRT Homosapiens 6 Leu Leu Asp Val Pro Thr Ala Ala Val 1 5 7 6 PRT Homo sapiens 7Ser Pro Ser Val Asp Lys 1 5 8 4 PRT Homo sapiens 8 Ala Arg Glu Ala 1 9 4PRT Homo sapiens 9 Gly Arg Asp Val 1

What is claimed is:
 1. A peptide consisting of an amino acid sequenceSPSVDKARAEL (SEQ ID NO:1) or FIDSYICQV (SEQ ID NO:2) wherein the peptideis immunologically recognizable as a T cell epitope of the minorhistocompatibility antigen polypeptide H-Y.
 2. A preparation, comprisinga peptide according to claim 1 and a pharmaceutically acceptableexcipient.
 3. A preparation according to claim 2, wherein thepreparation is a vaccine capable of inducing an immune response to theminor histocompatibility antigen H-Y.